6-beta-hydroxy-17 alpha-methyltestosterone, its esters and process



Patented Aug. 17, 1954 G-BETA- HYDROXY- 17 ALPHA-METHYL- ,TESTOSTERONE,ITS ESTERS AND PROCESS Herbert 0. Murray, Hickory Corners, and Darcy H.Peterson, Kalamazoo, Mich., assignors to The Upjohn Company, Kalamazoo,Mich., a corporation of Michigan No Drawing. Application January 15,1953,

Serial No. 331,493

9 Claims.

This invention relates to steroids and more particularly to certaintestosterone derivatives namely fip-hydroxy-l'la-methyltestosterone andits esters represented by the following formula:

wherein R is hydrogen or the acid radical of an aliphatic orcarb'ooyclic acid.

It is an object of this invention to provide the novelGp-hydroxy-l7a-methyltestosterone and its esters, and a process ofpreparing the same. Other objects will be apparent to those skilled inthe art to which this invention pertains.

The novel compounds of the present invention may be prepared froml'la-methyltestosterone by the oxygenating action of a culture offungus, particularly of the order Mucorales and the species Rhizopusrefleatus as described in application S. N. 297,242, filed July 5, 1952,of which this is a continuation-in-part.

Esterification may be accomplished by admixinfifi-hydroxy-17a-methyltestosterone with an acylating agent such as, forexample, ketene, a

ketene of a selected acid, an acid, acid chloride or acid anhydride, or.other known acylating agent, usually in a solvent such as, for example,pyridine or the like, or an inert solvent, including solvents likebenzene, toluene, ether, and the like, for example, and heated at atemperature between about zero degrees centigrade andthe boiling pointof the reaction mixture usually about room temperature, for a periodbetween about a half hour and about 96 hours. The time of reaction aswell as the temperature at which the reaction is carried out, theacylating agent, and the ratio of reactants may be varied. The reactionmixture is suitably poured into ice or cold water, the product collectedin an appropriate solvent which is thereafter washed with successiveportions of a mildly basic solution and water to obtain a solution ofthe product which is essentially neutral.

In some instances, the product may crystallize from the reactionmixture, in which case it may be advantageous to separate the product byfiltration or other means, wash with water, and" thereafter purify byconventional means, such as, for example, by recrystallization from asuitable solvent or by chromatographic purification, as deemednecessary.

The following examples are illustrative of the process and products ofthe present invention, but are not to be construed as limiting.

Example 1.--6 3 -hydroxy-1 7 a-methyltestosterone A medium was preparedof twenty grams of Edamine enzymatic digest of lactalbumin, three gramsof corn steep liquor and fifty grams of technical dextrose diluted toone liter with tap water and adjusted to a pH of 4.3 to 415. Twelveliters of this sterilized medium was inoculated with Rhizopus nigrz'cansminus strain, American Type Culture. Collection Number 6227?), and in..

cubated for 24 hours at a temperature of 28 degrees centigrade using arate of aeration and stirring such that the oxygen uptake was 6.3 to '7millimoles per hour per liter of NazSOs according to the method ofCooper, Fernstrom and Miller, Ind. Eng. Chem, 36, 504 (1944). To thismedium containing a 24-hour growth of Rhizopus m'gricans minus strainwas added six grams of l'la-methyltestosterone in milliliters ofabsolute ethanol to provide a suspension of the steroid in the culture.After an additional 24- hour period of incubation under the sameconditions of temperature and aeration, the beer and mycelium wereextracted The mycelium was filtered, washed twice, each time with avolume of acetone approximately equal to the volume of the mycelium andextracted twice, each time with a volume of methylene chlorideapproximately equal to the volume of the mycelium. The acetone andmethylene chloride extracts including solvent were added to the beerfiltrate. The mixed extracts and beer filtrate were extractedsuccessively with two one-half by volume portions of methylene chlorideand then withtwo one-fourth by volume portions of methylene chloride.The combined methylene chloride extracts were washed with two one-tenthby volume portions of a two percent aqueous solution of sodiumbicarbonate and then with two onetenth by volume portions of water.After drying the methylene chloride with about three to five grams ofanhydrous sodium sulfate per liter of solvent and filtering, the solventwas removed by distillation. The extractives obtained upon evaporationof the methylene chloride solvent were taken up in benzene to leave aresidue of benzene insoluble crystals. These were washed with additionalbenzene to give 550 milligrams of white crystals. Recrystallization froma mixture of equal parts of ethyl acetate and acetone gave 500milligrams of crystals melting at 220 to 235 degrees centigrade. Thesewere redissolved in three milliliters of methanol and ether was added tocrystallization. The resulting crystals of 6B hydroxy 17amethyltestosterone Weighed 212 milligrams and had a melting point of 247to 252 degrees centigrade. Infrared and ultraviolet spectra verified thestructure.

Example 2.-6 8acetoxy-17amethyltestosterone To fifty milligrams ofGB-hYdIQXYd'Za-methlltestosterone dissolved in one milliliter of bariumoxide dried pyridine was added one milliliter of a solution of 0.5milliliter of redistilled acetic anhydride diluted to ten milliliterswith dry pyridine and the reaction mixture maintained at roomtemperature for sixteen hours. The reaction mixture was then diluted tofifty milliliters and thereafter placed in a refrigerator for 24 hours.The thus-produced crystalline precipitate of 65- acetoxy 17mmethyltestosterone was filtered, washed with four one-milliliterportions of ice water and thereafter dried in vacuum at sixty degreescentigrade. The product was dissolved in 1.5 milliliters of methanol,the resulting solution filtered and thereafter diluted with 0.5milliliter of water whereafter there was precipitated crystals whichwere filtered, washed with three one-milliliter portions of ice waterand thereafter dried at seventy degrees centigrade to yieldfifi-acetoxy-Fla-methyltestosterone.

Example 3.-6p-jormyloxy-17a-methyltesto sterone In the same manner asExample 2, 6B-formyloxy-I'M-methyltestosterone is prepared by reactingdB-hydroxy-Fla-methyltestosterone with an excess of formic acid.

Example 4.6p-propiomyZo:ny-1 7 iii-methyltestosterone In the same manneras Example 2, 65-mopionyloxy-Fla-methyltestosterone is prepared byreacting 65-hydroxy-Fla-methyltestosterone with propionic anhydride inpyridine.

Example 5 .-6;8- 3-cycZopentyZ) propionylomy-lh methyltestosterone Inthe same manner as Example 2, SB-(fi-cyclopentyl) propionyloxy-l'lu.methyltestosterone is prepared by reacting 6,8-hydroxy-17a-methyltestosterone with B-cyclopentylpropionyl chloride in pyridine.

Example 6.6fl-benzoxy17a-methyltestosterone In the same manner asExample 2, GB-benzoxy- 17oc-lll6thY1t8St0St6I'0I18 is prepared byreacting fifl-hydroxy-lla-methyltestosterone with benzoyl chloride inpyridine.

Example 7 .6 -iceto-1 7 a-methyltestosterone A solution of 100milligrams of (SB-hydroxy- I'M-methyltestosterone in two milliliters ofglacial acetic acid was mixed with a' solution of 25 milligrams ofchromium trioxide in two milliliters of ninety percent acetic acid andmaintained at room temperature for five hours. ten milliliters ofmethanol was added to the mixture which was subsequently concentratedunder vacuum to remove most of the acetic acid and methanol. The residuewas mixed with 25 milli- Then liters of water and extracted with twofiftymilliliter portions of a mixture of five parts of ether and onepart of methylene chloride. The combined extracts were washed twice withtenmilliliter portions of five percent sodium bicarbonate solution andthree times with ten-milliliter portions of water. After drying overanhydrous sodium sulfate, the solvent was evaporated to leave acrystalline residue. Recrystallization twice from aqueous methanolyielded androgenic 6-keto-l'la-methyltestosterone.

In-a similar manner, other esters offifi-hydroxy-llei-methyltestosterone are prepared according to acylationprocedures, as illustrated above or by reaction with ketene, ketenes ofselected acids, selected acids, acid anhydrides, or acid chlorides, inan organic solvent such as pyridine or the like. Representative estersof 6/3-hydroxy 17a methyltestosterone thus-prepared include one to eightcarbon atom carboxylic acid acyloxy esters of saturated orunsaturated,aliphatic, carbocyclic, or cycloaliphatic,

aryl, arylalkyl, alkaryl, mono, di or polycarboxylic acids having lessthan nine carbon atoms and.

which form ester groups such as, for example, formyloxy, acetoxy,propionyloxy, dimethylacetoxy, trimethylacetoxy, butyryloxy, valeryloxy,hexanoyloxy, heptanoyloxy, octanoyloxy, benzoxy, phenylacetoxy,toluoyloxy, cyclopentylformyloxy, a and ,B-cyclopentylpropionyloxy,acrylyloxy, cyclohexylformyloxy, the half and diesters of polybasicacids, such as malonic, maleic, succinic, glutaric and adipic acids, andthe like. The acids may also contain non-interfering substituents, suchas mono or poly halo, chloro, bromo, hydroxy, methoxy, and the like ifdesired.

The Gfi-hydroxy-Nix-methyltestosterone and. its carboxylic acid estersare useful chemical intermediates and have pharmacological activity perse. They exhibit androgenic, progestational and anaesthetic activity.

It is to be understood that this invention is not to be limited to theexact details of operation or exact compounds shown and described asobvious modifications and equivalents will be apparent to one skilled inthe art and the invention is therefore to be limited only by the scopeof the appended claims.

wherein R is selected from the radicals consisting of hydrogen andhydrocarboncarboxylic acyl radical containing less than nine carbonatoms.

2. GB-hydroxy-Fla-methyltestosterone,

3. 6,8-acyloxy-I'M-methyltestosterone wherein the acyl radical is thatof an aliphatic hydrocarbon carboxylic acid containing less than ninecarbon atoms.

4. (SB-acyloxy-Fla-methyltestosterone wherein the acyl radical isthat ofa carbocyclic hydrocarbon carboxylic acid containing less than ninecarbonatoms.

5. A 6,8-acyloxy-l'la-methyltestosterone represented by the structuralformula:

RCO

wherein R is a hydrocarbon radical containing less than eight carbonatoms.

6. 6,9-acetoxy-l'la-methyltestosterone.

7. fifi-propionyloxy-l'la-methyltestosterone.

8. Gfl-(B-cyclopentyl)propionyloxy I'M-methyltestosterone.

6 9. A process of preparing a compound represented by the graphicalformula:

N 0 references cited.

1. A COMPOUND REPRESENTED BY THE GRAPHICAL FORMULA: